LOCUS       sequence-221665-        8576 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  allelic exchange vector with GFP merodiploid tracker, 
            temperature-sensitive origin of replication, and TetR-controlled 
            kill switch.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8576)
  AUTHORS   Wiles TJ, Wall ES, Schlomann BH, Hay EA, Parthasarathy R, Guillemin 
            K
  TITLE     Modernized Tools for Streamlined Genetic Manipulation and 
            Comparative Study of Wild and Diverse Proteobacterial Lineages.
  JOURNAL   MBio. 2018 Oct 9;9(5). pii: mBio.01877-18. doi: 
            10.1128/mBio.01877-18.
  PUBMED    30301859
REFERENCE   2  (bases 1 to 8576)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "MBio."; 
            date: "2018-10-9"; volume: "9(5). pii: mBio.01877-18. doi"; pages: "
            10.1128/mBio.01877-18"
COMMENT     Sequence Label: pAX2
FEATURES             Location/Qualifiers
     source          1..8576
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            370..478
                     /note="incP origin of transfer"
     promoter        join(742..747,748..764,765..770)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             959..1492
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        1606..1708
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(1775..1794)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     RBS             2428..2439
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2446..3159
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2476..2504)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3099..3119
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      3202..3296
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      3399..3485
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     RBS             3932..3940
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     RBS             4057..4079
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4108..4126
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4133..4151
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        join(4172..4177,4178..4193,4194..4200)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             4237..4860
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(4310..4329)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5078..5097)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5197..5219
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5257..5275)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5343..5447
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5448..5516,5517..6308)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5666..5685)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7356..7578
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             7626..8576
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
ORIGIN
        1 ggttcttatg gctcttgtat ctatcagtga agcatcaaga ctaacaaaca aaagtagaac
       61 aactgttcac cgttacatat caaagggaaa actgtccata cccatgtaaa acttaaaacc
      121 ttagaggcta tttaagttgc tgatttatat taattttatt gttcaaacat gagagcttag
      181 tacgtgaaac atgagagctt agtacgttag ccatgagagc ttagtacgtt agccatgagg
      241 gtttagttcg ttaaacatga gagcttagta cgttaaactt gagagcttag tacgtgaaac
      301 atgagagctt agtacgtact atcaacaggt tgaactgccc atgttctttc ctgcgttatc
      361 agagcttatc ggccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaata
      421 agggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta tcctgcccgg
      481 ctgacgccgt tggatacacc aaggaaagtc tacacgaacc ctttggcaaa atcctgtata
      541 tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata atgaccccga agcagggtta
      601 tgcagcggaa agtatacctt aaggaatccc ctgataccgc tcgccgcagc cgaacgaccg
      661 agcgcagcgg gtcagtgagc gaggaagcgg aagagaattc caagatcccc tgattccctt
      721 tgtcaacagc aatggatcga attgacataa gcctgttcgg ttcgtaaact gtaatgcaag
      781 tagcgtatgc gctcacgcaa ctggtccaga accttgaccg aacgcagcgg tggtaacggc
      841 gcagtggcgg ttttcatggc ttgttatgac tgtttttttg tacagtctat gcctcgggca
      901 tccaagcagc aagcgcgtta cgccgtgggt cgatgtttga tgttatggag cagcaacgat
      961 gttacgcagc agcaacgatg ttacgcagca gggcagtcgc cctaaaacaa agttaggtgg
     1021 ctcaagtatg ggcatcattc gcacatgtag gctcggccct gaccaagtca aatccatgcg
     1081 ggctgctctt gatcttttcg gtcgtgagtt cggagacgta gccacctact cccaacatca
     1141 gccggactcc gattacctcg ggaacttgct ccgtagtaag acattcatcg cgcttgctgc
     1201 cttcgaccaa gaagcggttg ttggcgctct cgcggcttac gttctgccca agtttgagca
     1261 gccgcgtagt gagatctata tctatgatct cgcagtctcc ggcgagcacc ggaggcaggg
     1321 cattgccacc gcgctcatca atctcctcaa gcatgaggcc aacgcgcttg gtgcttatgt
     1381 gatctacgtg caagcagatt acggtgacga tcccgcagtg gctctctata caaagttggg
     1441 catacgggaa gaagtgatgc actttgatat cgacccaagt accgccacct aactctagac
     1501 aatatcgata cctgtgacgg aagatcactt cgcagaataa ataaatcctg gtgtccctgt
     1561 tgataccggg aagccctggg ccaacttttg gcgaaaatga gacgttgatc ggcacgtaag
     1621 aggttccaac tttcaccata atgaaataag atcactaccg ggcgtatttt ttgagttgtc
     1681 gagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt
     1741 tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg
     1801 tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa
     1861 taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc
     1921 ggaattacgt atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg
     1981 ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga
     2041 cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct
     2101 ggcctatttc cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt
     2161 gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt
     2221 caccatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt
     2281 tcatcatgcc gtttgtgatg gcttccatgt cggcagatgc ttaatgaata caacagtact
     2341 gcgatgaact agtctttggc agtttattct tgacatgtag tgagggggct ggtataatca
     2401 catagtactg ttcgggtacc tagaattaaa gaggagaaat taagcatgcg taaaggagaa
     2461 gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac
     2521 aaattttctg tcagtggaga gggtgaaggt gatgcaacat acggaaaact tacccttaaa
     2581 tttatttgca ctactggaaa actacctgtt ccatggccaa cacttgtcac tactttcggt
     2641 tatggtgttc aatgctttgc gagataccca gatcatatga aacagcatga ctttttcaag
     2701 agtgccatgc ccgaaggtta tgtacaggaa agaactatat ttttcaaaga tgacgggaac
     2761 tacaagacac gtgctgaagt caagtttgaa ggtgataccc ttgttaatag aatcgagtta
     2821 aaaggtattg attttaaaga agatggaaac attcttggac acaaattgga atacaactat
     2881 aactcacaca atgtatacat catggcagac aaacaaaaga atggaatcaa agttaacttc
     2941 aaaattagac acaacattga agatggaagc gttcaactag cagaccatta tcaacaaaat
     3001 actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc cacacaatct
     3061 gccctttcga aagatcccaa cgaaaagaga gaccacatgg tccttcttga gtttgtaaca
     3121 gctgctggga ttacacatgg catggatgaa ctatacaaat aagcgagctc gtttaaacgc
     3181 tagcttaatt agctgagctt ggactcctgt tgatagatcc agtaatgacc tcagaactcc
     3241 atctggattt gttcagaacg ctcggttgcc gccgggcgtt ttttattggt gagaatccaa
     3301 gctagactgc gatgagtggc agggcggggc gtaatttttt taaggcagtt attggtgccc
     3361 ttaaacgcct ggggtaatga ctctctagct tgaggcatca aataaaacga aaggctcagt
     3421 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
     3481 caaatccgcc gctaggagtc gcgagtttaa accccggggt cgaccagact cgagaaatgc
     3541 gcggggaaaa tccccgcgct tgcccttaat taagtattac ttcaggtatt tcagaacagc
     3601 atcaagcagt tgcagtgctg caacaatgag tttgaggata agaatggcga tatccaccag
     3661 gttcatacgc gtctcctggc gccgtcgacc taaaagagag aaaaaagtaa tgccacaggc
     3721 agactcattg gccaggttat tccgaccagg aatgcactaa gtaaacgaat atgtgttttc
     3781 tcatgtgaga taaaccagat aatgacaaag gatatgttca caccaatcac ataaaaaatt
     3841 aatattttag agaatagtgc cattttttat gttcttctta tccttcctgt cgactcgcga
     3901 ttacctggac gtgcaggcca tgagcgcagc aacctcctta tcaccgtccc ggaaccgcag
     3961 ttcgtagagc gtttgtcggg tcaggagtgt gaatgtcaga atcgtaatgc agataatgag
     4021 cagacacacc accagagggt tgtgcttcat agcctttctc cttcttaaag ttaaacaaaa
     4081 ttatttctag aagctttgtg ctcagtatct ctatcactga tagggatgtc aatctctatc
     4141 actgataggg aggccggccc ctagggagct gttgacaatt aatcatcggc tcgtataatg
     4201 tgtggaactc aagaacaata atctaggtat tacatcatgt ctagattaga taaaagtaaa
     4261 gtgattaaca gcgcattaga gctgcttaat gaggtcggaa tcgaaggttt aacaacccgt
     4321 aaactcgccc agaagctagg tgtagagcag cctacattgt attggcatgt aaaaaataag
     4381 cgggctttgc tcgacgcctt agccattgag atgttagata ggcaccatac tcacttttgc
     4441 cctttagaag gggaaagctg gcaagatttt ttacgtaata acgctaaaag ttttagatgt
     4501 gctttactaa gtcatcgcga tggagcaaaa gtacatttag gtacacggcc tacagaaaaa
     4561 cagtatgaaa ctctcgaaaa tcaattagcc tttttatgcc aacaaggttt ttcactagag
     4621 aatgcattat atgcactcag cgctgtgggg cattttactt taggttgcgt attggaagat
     4681 caagagcatc aagtcgctaa agaagaaagg gaaacaccta ctactgatag tatgccgcca
     4741 ttattacgac aagctatcga attatttgat caccaaggtg cagagccagc cttcttattc
     4801 ggccttgaat tgatcatatg cggattagaa aaacaactta aatgtgaaag tgggtcttaa
     4861 ctgcagcccg cttacgcagg gcatccattt attactcaac cgtaaccgat tttgccaggt
     4921 tacgcggctg gtcctctagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     4981 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc
     5041 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat
     5101 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     5161 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     5221 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     5281 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     5341 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5401 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     5461 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5521 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5581 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5641 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5701 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5761 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5821 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5881 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5941 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     6001 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     6061 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6121 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     6181 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6241 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6301 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     6361 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     6421 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6481 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6541 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6601 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     6661 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gccggttgtc
     6721 agccgttaag tgttcctgtg tcactcaaaa ttgctttgag aggctctaag ggcttctcag
     6781 tgcgttacat ccctggcttg ttgtccacaa ccgttaaacc ttaaaagctc atgaccaaaa
     6841 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgttgatgat accgctgcct
     6901 tactgggtgc attagccagt ctgaatgacc tgtcacggga taatccgaag tggtcagact
     6961 ggaaaatcag agggcaggaa ctgctgaaca gcaaaaagtc agatagcacc acatagcaga
     7021 cccgccataa aacgccctga gaagcccgtg acgggctttt cttgtattat gggtagtttc
     7081 cttgcatgaa tccataaaag gcgcctgtag tgccatttac ccccattcac tgccagagcc
     7141 gtgagcgcag cgaactgaat gtcacgaaaa agacagcgac tcaggtgcct gatggtcgga
     7201 gacaaaagga atattcagcg atttgcccga ttgcggccgc aaccgagctt gcgagggtgc
     7261 tacttaagcc tttagggttt taaggtctgt tttgtagagg agcaaacagc gtttgcgaca
     7321 tccttttgta atactgcgga actgactaaa gtagtgagtt atacacaggg ctgggatcta
     7381 ttctttttat ctttttttat tctttcttta ttctataaat tataaccact tgaatataaa
     7441 caaaaaaaac acacaaaggt ctagcggaat ttacagaggg tctagcagaa tttacaagtt
     7501 ttccagcaaa ggtctagcag aatttacaga tacccacaac tcaaaggaaa aggactagta
     7561 attatcattg actagcccat ctcaattggt atagtgatta aaatcaccta gaccaattga
     7621 gatgtatgtc tgaattagtt gttttcaaag caaatgaact agcgattagt cgctatgact
     7681 taacggagca tgaaaccaag ctaattttat gctgtgtggc actactcaac cccacgattg
     7741 aaaaccctac aaggaaagaa cggacggtat cgttcactta taaccaatac gctcagatga
     7801 tgaacatcag tagggaaaat gcttatggtg tattagctaa agcaaccaga gagctgatga
     7861 cgagaactgt ggaaatcagg aatcctttgg ttaaaggctt tgagattttc cagtggacaa
     7921 actatgccaa gttctcaagc gaaaaattag aattagtttt tagtgaagag atattgcctt
     7981 atcttttcca gttaaaaaaa ttcataaaat ataatctgga acatgttaag tcttttgaaa
     8041 acaaatactc tatgaggatt tatgagtggt tattaaaaga actaacacaa aagaaaactc
     8101 acaaggcaaa tatagagatt agccttgatg aatttaagtt catgttaatg cttgaaaata
     8161 actaccatga gtttaaaagg cttaaccaat gggttttgaa accaataagt aaagatttaa
     8221 acacttacag caatatgaaa ttggtggttg ataagcgagg ccgcccgact gatacgttga
     8281 ttttccaagt tgaactagat agacaaatgg atctcgtaac cgaacttgag aacaaccaga
     8341 taaaaatgaa tggtgacaaa ataccaacaa ccattacatc agattcctac ctacgtaacg
     8401 gactaagaaa aacactacac gatgctttaa ctgcaaaaat tcagctcacc agttttgagg
     8461 caaaattttt gagtgacatg caaagtaagt atgatctcaa tggttcgttc tcatggctca
     8521 cgcaaaaaca acgaaccaca ctagagaaca tactggctaa atacggaagg atctga
//
